Evaluations of Shorter Exposures of Contact Lens Cleaning Solutions ...

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, May 2011, p. 2265–2275 0066-4804/11/$12.00 doi:10.1128/AAC.00634-09 Copyright © 2011, American Society for Microbiology. All Rights Reserved.

Vol. 55, No. 5

Evaluations of Shorter Exposures of Contact Lens Cleaning Solutions against Fusarium oxysporum Species Complex and Fusarium solani Species Complex To Simulate Inappropriate Usage䌤 Rama Ramani† and Vishnu Chaturvedi* Mycology Laboratory, Wadsworth Center, New York State Department of Health, and Department of Biomedical Sciences, School of Public Health, University at Albany, Albany, New York Received 11 May 2009/Returned for modification 27 July 2009/Accepted 30 January 2011

An outbreak of Fusarium keratitis in contact lens users resulted in withdrawal of ReNu with MoistureLoc solution, although the exact cause of the outbreak remains enigmatic. We evaluated current and discontinued multipurpose cleaning solutions (MPSs; MoistureLoc, Equate, MultiPlus, and OptiFree Express) against plankton- and biofilm-derived cells of Fusarium oxysporum species complex (FOSC) and F. solani species complex (FSSC). The methods included a traditional assay based on CFU counts and a novel flow cytometry (FC) assay based on percent cell subpopulation (PCS) stained with two fluorochromes (Sytox Red and 5-chloromethylfluorescein diacetate). The tests were done with the respective manufacturers’ recommended cleaning regimens (240 to 360 min) and under shorter exposures (15 to 60 min) to simulate inappropriate usage by the customers. FC assay measured PCS, which was available rapidly, in 5 to 7 h, whereas 24 to 48 h was needed for CFU counts, and there was good correlation between the two methods (r2 ⴝ 0.97). FC assays allowed identification of injured fungal cells, which are likely to be missed with growth assays. In general, a time- and inoculum-dependent survival pattern was seen for both FOSC and FSSC cells, and biofilm-derived cells were more resistant than plankton-derived cells. MultiPlus and Equate produced 100% sterilization of fungi even under shorter exposures. However, biofilm FOSC and FSSC cells survived for up to 4 h in MoistureLoc solution and up to 6 h in OptiFree Express solution under shorter exposure times. This finding was enigmatic, as OptiFree Express is not associated with any outbreak of Fusarium keratitis. This study provides additional support for possible roles that improper lens cleaning regimens and fungal biofilms could play as predisposing factors for Fusarium keratitis. factor in recent infections (11). Another group of investigators reported a more expanded evaluation involving corneal isolates from all parts of the United States and found a high degree of phylogenetic diversity in fungal isolates (22). This work was based on an earlier study by Zhang et al., who showed that FSSC strains associated with human and animal infections have their origins from multiple sources in the environment (33). A number of laboratories have recently reported the findings of experimental studies seeking to account for the outbreak related to use of ML (1, 7, 11, 13, 14, 18, 19, 27, 34). Imamura et al. concluded that the formation of biofilms on various types of contact lenses and the consequent reduced susceptibility to lens cleaning solutions played a role in the outbreak of Fusarium keratitis (14). Zhang et al. suggested that the outbreak was due to the survival of FSSC and FOSC cells on the plastic surfaces of contact lens cases that contained residual ML solution in the form of stressed ML films (34). Bullock et al. suggested that exposure of ML to relatively high temperatures caused decreased fungicidal activity and that this contributed to the Fusarium keratitis outbreak (7). Ahearn et al. described the attachment of F. solani to contact lenses and subsequent failure to manually clean these lenses to explain the increased incidence of Fusarium keratitis (2). Furthermore, the ML manufacturer has also reported that ML showed reduced biocidal activity under conditions that stimulated noncompliance in a number of ways, including evaporation of the solution, but not under the conditions recommended for its use (19). Impor-

Fusarium keratitis associated with use of contact lens cleaning solution was reported from Singapore, with an upsurge of cases from March 2005 to May 2006; 94% of the patients had reportedly used ReNu MoistureLoc (ML) contact lens cleaning solution (16). That report was followed in rapid succession by a number of cases described from various parts of the United States. Chang et al. reported that the outbreak of Fusarium keratitis was associated with use of ReNu with MoistureLoc contact lens solution; they recommended that lens wearers not use this contact lens solution (8). An epidemiological investigation in Hong Kong also reported a significant association between Fusarium keratitis cases and the use of ML solution (20). However, the source of the outbreak remained inconclusive, since Fusarium was not isolated from the factory or from unopened bottles of ML (8). We were involved in laboratory investigations of the very first few cases in the United States, which originated from the New York and New Jersey area with Fusarium oxysporum species complex (FOSC) (13). Previously, we showed that temporary survival of FOSC and F. solani species complex (FSSC) in ML due to an improper lens cleaning regimen could be a * Corresponding author. Mailing address: Mycology Laboratory, Wadsworth Center, New York State Department of Health, 120 New Scotland Ave., Albany, NY 12201-2002. Phone: (518) 474-4177. Fax: (518) 486-7971. E-mail: [email protected]. † Present address: Laboratory for Viral Diseases, Wadsworth Center, Albany, NY. 䌤 Published ahead of print on 7 February 2011. 2265

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ANTIMICROB. AGENTS CHEMOTHER.

FIG. 1. Biofilm architecture of FOSC and FSSC developed in a flow cell device (Stovall [Greensboro, NC] flow cell). The biofilms were stained with CMFDA and Sytox Red for 1 h and analyzed by confocal scanning laser microscope (Leica [Exton, PA] TCS SP5). (A) Top-view architecture of FOSC (MYC-158-06) biofilm displaying compact, interwoven, viable hyphae (green) with few dead fragments (red); (B) top-view architecture of FSSC (MYC23-06) biofilm displaying interwoven, relatively less compact viable hyphae, microconidia, intercalary chlamydospores (green), and a few dead cells (red); (C) FOSC biofilm displaying a depth of about 25 ␮m; (D) FSSC biofilm displaying a depth of 34 ␮m. Bar ⫽ 150 ␮m.

tantly, it is still not clear what the standard compliance condition is, since a recent study reported that “inconsistent and inadequate contact lens and lens case hygiene recommendations remain prevalent among various advisory bodies” (31). Overall, there are still many unanswered questions about the pathogenesis of Fusarium keratitis in recent outbreaks (29). Previous studies have measured fungal viability by traditional fungal growth assay using CFU counts or by calorimetry of biofilms (1, 14, 34). The current International Organization for Standards (ISO) protocol describes the use of F. solani ATCC 36031; this strain is susceptible, while the clinical and environmental fungal isolates are significantly resistant to commercially available lens cleaning solutions (5, 12). However, fungal cells could exist in various stages of viability when they are exposed to a deleterious compound. Our laboratory has published many reports on the utility of flow cytometry (FC) to define subpopulations of fungal pathogens that are variously damaged upon exposure to antifungal drugs (9, 25, 26). Therefore, we decided to use FC to characterize the effects of multipurpose cleaning solutions (MPSs) on plankton- and biofilmderived cells of Fusarium species and to correlate the results with those of traditional CFU assay.

MATERIALS AND METHODS Fungal isolates and lens cleaning solutions. Three isolates each of FOSC (accession numbers MYC-158-06, MYC-159-06, and MYC-398-06) and FSSC (accession numbers MYC-23-06, MYC-288-06, and MYC-299-06) from the Wadsworth Center Mycology Laboratory Fungus Culture Collection were used. These isolates were obtained from the corneas of keratitis patients who wore contact lenses; these isolates were part of our previous study (11). These isolates were stored in sterile water at room temperature and also in 15% sterile glycerol under liquid nitrogen. Bottles of ReNu with ML (Bausch & Lomb, Rochester, NY) were purchased from local pharmacies, before the recall by the manufacturer; these solutions were used for the experiments before the printed expiry dates. Other MPSs tested were Equate (EQ; Wal-Mart Inc., Bentonville, AK), MultiPlus (MP; Bausch & Lomb, Rochester, NY), and OptiFree Express (OP; Alcon Laboratories, Inc., Fort Worth, TX); these were purchased from local pharmacies and stored according to the manufacturers’ recommendations. Biofilms. It has been suggested that the recent Fusarium keratitis outbreak was caused by formation of fungal biofilms on contact lenses and/or lens cases (14). Hence, we decided to include biofilm-derived cells in biofouling experiments. FSSC or FOSC biofilms were grown in continuous-flow chambers, using RPMI 1640 broth (Invitrogen, Carlsbad, CA). For the purpose of biofilm formation, cell suspensions from 4- to 5-day-old colonies were suspended in sterile saline. Cells were counted to 1 ⫻ 105 cells/ml, and 100 ␮l of this conidial suspension was inoculated into a flow chamber (Stovall [Greensboro, NC] flow cell) and incubated at room temperature for 2 h with the coverslip facing down to promote adherence. Subsequently, the chamber was inverted (coverslip up), and the medium (RPMI 1640 broth) was pumped through the chamber at a flow rate of 180 ␮l per min for 72 h. At the end of the experiment, the coverslips were lifted and stained with 5-chloromethylfluorescein diacetate (CMFDA; 1 ␮l of a

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FIG. 2. FC parameters for differentiating live-dead cells of FSSC (MYC- 23-06). The two fluorochromes used were CMFDA (emitting green fluorescence) and Sytox Red (emitting red fluorescence). This combination had minimal emission overlap and needed no spectral compensation in recording of results. Plankton-derived cells were prepared from growth on potato dextrose agar slants, and biofilm-derived cells were prepared from cultures grown in a flow cell device (Stovall [Greensboro, NC] flow cell). For dead cell preparations, a cell suspension was incubated with an equal volume of methanol. Viability was checked by the trypan blue exclusion test. The cell suspensions were analyzed in a FACSCalibur apparatus (Becton Dickinson, Lincoln Park, NJ). Live cells were identified in the FL1 channel (x axis), while dead cells were identified in the FL4 channel (y axis). Electronic gates were set up in dot plots of FSC versus SSC to capture the target cell population and to exclude cell debris (details not shown). (A) Density plot of 100% live plankton-derived cells depicting green fluorescence in lower right quadrant; (B) density plot of 100% dead plankton-derived cells depicting red fluorescence in upper left quadrant; (C) density plot of 100% live biofilm-derived cells depicting green fluorescence in lower right quadrant; (D) density plot of 100% dead biofilm-derived cells depicting red fluorescence in upper left quadrant. A similar experimental setup was used for FOSC live-dead cells (details not shown).

1-mg/ml stock solution) and Sytox Red (1 ␮l of a 5 ␮M stock solution) in sterile petri plates for 1 h in the dark. After incubation, the biofilms were washed with sterile Milli-Q water and observed by confocal scanning laser microscopy (CSLM; Leica [Exton, PA] TCS SP5 confocal microscope). FC assay for viability. FC was calibrated daily by use of standard beads (Becton Dickinson Inc., Lincoln Park, NJ) and by use of live and dead fungal cells (26). Live inoculum was prepared from plankton-derived cells as described for the standard growth assays. For dead cells, the cell suspension was incubated with an equal volume of methanol for 30 min and centrifuged at 5,000 rpm for 5 min; the supernatant was then removed and the pellet was resuspended using sterile, deionized water to constitute the original volume of cell suspension. Viability was checked by the trypan blue exclusion test. Several fluorochromes, such as propidium iodide (PI), 3⬘-dipentyloxacarbocyanine iodide (DiOC5), 2-choro-4-[2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-methylidene]-1 phenylquinolinium iodide (FUN-1), fluorescein diacetate (FDA), Syto-9, ethidium bromide, rhodamine 123, Sytox Orange, and calcein A with aqua fluorescence, were tried in various combinations (Molecular Probes/Invitrogen, Carlsbad, CA). Since the CMFDA and Sytox Red combination reliably differentiated between

live and dead cells, these fluorochromes were used in subsequent experiments. CMFDA localizes in the cytoplasm of viable cells, emitting green fluorescence, and Sytox Red binds to nucleic acids in dead cells, emitting red fluorescence. Biofouling experiments (neutralization versus nonneutralization procedures). In the initial stages of this study, the neutralization steps and nonneutralization steps were carried out simultaneously for ML and MP using two strains each of FOSC and FSSC. Only plankton-derived cells were used, as follows: conidia and hyphal fragments from 4- to 5-day-old cultures from potato dextrose agar slants at 30°C were suspended in sterile distilled water (SDW). The conidia were counted with a hemocytometer and adjusted to a final density of 1 ⫻ 104 to 107 cells/ml in sterile distilled water. For the neutralization procedure, 10 ␮l of each fungal cell suspension was inoculated into 1.0 ml of MPS. The MPS-fungus suspension was incubated unstirred at 23°C to 25°C for various time intervals (15, 30, 60, and 240 or 360 min), 100-␮l aliquots were taken at the stipulated time, and 10 ␮l of each sample was mixed with 100 ␮l neutralizing broth (Dey-Engley broth) and incubated for 15 min at 23°C to 25°C. After 15 min, 110 ␮l was spread on the Sabouraud’s dextrose agar (SDA) plates and incubated for 2 to 3 days. For the nonneutralization procedure, 10 ␮l of each fungal cell suspension was inoc-

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TABLE 1. Results of biofouling experiments obtained with neutralization and nonneutralization tests with ML and MPa FOSC Solution and time period (min)

a

FSSC

Mean % killing ⫾ SD


Neutralization test

Nonneutralization test

P value

Neutralization test

Nonneutralization test

P value

ML 15 30 60 240

54 ⫾ 2.2 33 ⫾ 3.8 14 ⫾ 3.7 0

52 ⫾ 2.7 28 ⫾ 1.8 11 ⫾ 4.3 0

⬎0.05 ⬎0.05 ⬎0.05

31 ⫾ 2.5 20 ⫾ 3.5 12 ⫾ 3.2 0

32 ⫾ 2.0 24 ⫾ 2.1 11 ⫾ 3.9 0

⬎0.05 ⬎0.05 ⬎0.05

MP 15 30 60 240

0 0 0 0

0 0 0 0

0 0 0 0

0 0 0 0

Two strains each of FOSC and FSSC were tested.

ulated into 1.0 ml of MPS. The MPS-fungus suspension was incubated unstirred at 23°C to 25°C for various time intervals (15, 30, 60, and 240 or 360 min), and 100-␮l aliquots were taken and spread on SDA plates and incubated for 2 to 3 days. At the end of incubation, the colonies were counted for each time period, and the percent killing was compared between the neutralization and nonneutralization procedures. Data were collected from three independent experiments. Biofouling experiments (glass tubes versus lens cases). We earlier described that there was no appreciable difference between glass tubes and lens cases when they were used as containers for interactions (11). This was again tested in this study to verify that observation. Briefly, the conidia and hyphal fragments from 4- to 5-day-old cultures from slants of potato dextrose agar at 30°C were suspended in SDW, counted with a hemocytometer, and adjusted to a final density of 1 ⫻ 104 to 107 cells/ml. Ten microliters of the respective fungal cell suspension was inoculated into 1.0 ml of ML in glass tubes or lens cases. The suspension was incubated unstirred at 23°C to 25°C for various time intervals (15, 30, 60, and 240 or 360 min), and 100-␮l aliquots were taken, spread on SDA plates, and incubated for 2 to 3 days. Data were collected from three independent experiments. Biofouling experiments using plankton-derived cells (CFU method). Conidia and hyphal fragments from 4- to 5-day-old cultures of FOSC and FSSC on slants of potato dextrose agar at 30°C were suspended in SDW. The suspension mainly comprised conidia, with rare hyphal fragments. The conidia were counted with a hemocytometer and adjusted to a final density of 1 ⫻ 104 to 107 cells/ml in sterile distilled water. This simulated both low and high levels of fungal contaminants from plankton-derived cells. In the previous publication from our group, no appreciable differences in Fusarium survival rates in biofouling experiments were observed when glass tubes or contact lens storage cases were used (11). Hence, all incubations were done in glass tubes in the present study. Ten microliters of each fungal cell suspension was inoculated into 1.0 ml of MPS in a sterile glass test tube (20 by 150 mm). The MPS-fungus suspension was incubated unstirred at 23°C to 25°C for various time intervals (15, 30, 60, and 240 or 360 min), and 100-␮l aliquots were taken and spread on SDA plates. The positive controls included fungal inoculum suspended in SDW without addition of any MPS. The inoculated plates were incubated at 25°C for 48 to 72 h, and the numbers of fungal CFU were counted and photographed. Data were collected from three independent experiments. Biofouling experiments using biofilm-derived cells (CFU method). After 72 h, the biofilm-derived cells were suspended in SDW by scraping the growth with a sterile plastic inoculation loop. The hyphal fragments along with conidia were counted with a hemocytometer and adjusted to a final density of 1 ⫻ 104 to 107 cells/ml. This simulated both low and high levels of fungal contaminants. Ten microliters of each suspension was used to inoculate 1.0 ml of various MPSs in sterile glass test tubes (20 by 150 mm). Biofouling experiments were set up similarly to the experiments with the plankton-derived cells described earlier. Biofouling experiments (FC assay). Plankton- and biofilm-derived cells were counted with a hemocytometer and adjusted to a final density of 1 ⫻ 104 to 107 cells/ml. Ten microliters of each of these suspensions was used to inoculate 1.0 ml of MPS. The mix was incubated unstirred at 23°C to 25°C for various time intervals (15, 30, 60, and 240 or 360 min), and 200-␮l aliquots were then removed. Cells were then washed; two dyes, CMFDA and Sytox Red, were added; and the mixture was further incubated for 1 h and shaken at 10 rpm in the dark.

The cell suspensions were analyzed either in a flow cytometer (FACSCalibur; Becton Dickinson, Lincoln Park, NJ) or a fluorescence-activated cell sorter (FACS; Aria; Becton Dickinson, Lincoln Park, NJ). In FC, the percentages of the subpopulations (percent cell subpopulation [PCS]) were calculated after subtraction of the debris from the total cell counts. The live cell subpopulation correlated with CMFDA uptake (CMFDA positive [CMFDA⫹]/Sytox Red negative [Sytox Red⫺]), while the dead subpopulation correlated with Sytox Red uptake (CMFDA negative [CMFDA⫺]/Sytox Red positive [Sytox Red⫹]). Subpopulation cells stained with both fluorochromes (CMFDA⫹/Sytox Red⫹) were injured or stressed; presumably, the membrane and organelle damage in these cells was enough for the uptake of Sytox Red, yet the remainder of the viable metabolic activity allowed retention of CMFDA. The fourth subpopulation did not retain any fluorochromes (CMFDA⫺/Sytox Red⫺). This was either cell debris or cells with a loss of all activity and integrity. PCS for CMFDA⫹ alone of Fusarium cells treated with MPS was the FC measure of fungal cell viability. Various cell populations were collected from the Aria FACS apparatus, plated onto SDA plates, and incubated at 25°C to determine the numbers of CFU. Data were collected from three independent experiments. Data analysis. Regression analysis was performed with Microsoft Office Excel 2007 software to find the levels of correlation between the numbers of CFU and PCS data.

RESULTS Biofilm formation. The flow cell device allowed formation of uniform, compact biofilms of FOSC and FSSC. CSLM revealed that both FOSC and FSSC formed biofilms that were rich in hyphae and that had a homogeneous architecture. The biofilms were stained with two fluorochromes, because CMFDA stains live cells, while Sytox Red penetrates only dead cells or cells with a compromised cell membrane (Fig. 1). More details on dual fluorochromes are included in the next section on FC. The biofilms comprised mostly live cells highlighted with the green fluorescence of CMFDA; there were few dead or injured cells in the biofilms, indicated by the red fluorescence of Sytox Red (Fig. 1). The depth of the biofilm was calculated using CSLM, and the depths were 25 ␮m for FOSC and 34 ␮m for FSSC. FC assay for cell viability. Initially, we tested combinations of PI, ethidium bromide, rhodamine 123, or Sytox Orange with DiOC5, FUN-1, Syto-9, CMFDA, FDA, and calcein A with aqua fluorescence. These fluorochromes did not validate the live-dead cell populations in our hands due to one or more of the following reasons: although electronic color compensations were set on the instrument to reduce the spectral overlaps,

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TABLE 2. Results of biofouling experiments obtained with glass tubes and lens cases with ML and MPa FOSC Time period (min)

15 30 60 240 a


FSSC

a

Glass tubes

Lens cases

45 ⫾ 2.0 33 ⫾ 4.5 9 ⫾ 1.1 0

48 ⫾ 4.1 39 ⫾ 1.8 8 ⫾ 1.8 0

P value

⬎0.05 ⬎0.05 ⬎0.05

Mean % killing ⫾ SDa Glass tubes

Lens cases

36 ⫾ 1.6 22 ⫾ 4.4 8 ⫾ 2.6 0

39 ⫾ 3.2 25 ⫾ 3.9 11 ⫾ 2.9 0

P value

⬎0.05 ⬎0.05 ⬎0.05

Two strains each of FOSC and FSSC were tested.

there were significant spectral overlaps; the shift in log fluorescence was minimal as the probes either did not enter or did not leave the dead fungal cells completely; or only one probe showed a prominent fluorescence shift. However, the combination of CMFDA and Sytox Red showed a 2-log-scale shift in fluorescence between live and dead cells. We found that these two fluorescent dyes could differentiate between live and dead Fusarium cells. Thus, live cells stained with CMFDA produced fluorescence in the green channel, whereas dead cells stained with Sytox Red produced fluorescence in the red channel. These two probes were excited at different wavelengths: CMFDA at 488 nm and Sytox Red at 640 nm. Hence, the need for compensation and overlapping was minimal. The two fluorescent probes reproducibly differentiated between live and dead cell populations in the FACSCalibur apparatus (Fig. 2). Electronic gates were set up in dot plots of forward scatter

(FSC) versus side scatter (SSC) to capture the target cell population and to exclude cell debris (details not shown). Plankton-derived live cells of FSSC (MYC-23-06) were identified in the FL1 channel (green fluorescence; Fig. 2A, lower right quadrant), while dead cells were identified in the FL4 channel (red fluorescence; Fig. 2B, upper left quadrant). Biofilm-derived live cells of FSSC (MYC-23-06) were identified in the FL1 channel (green fluorescence; Fig. 2C, lower right quadrant), while dead cells were identified in the FL4 channel (red fluorescence; Fig. 2D, upper left quadrant). Biofouling experiments (neutralization versus nonneutralization). Results of neutralization and nonneutralization experiments obtained with ML and MP are summarized in Table 1. There were no significant differences with and without the use of neutralizing broth (P ⬎ 0.05). Since the neutralization and nonneutralization procedures did not reveal significant

FIG. 3. Summary of experimental biofouling studies with FOSC evaluated by CFU method. Fungal cells (1 ⫻ 105 cells/ml) were inoculated into ML and OP solutions, and aliquots were withdrawn at the indicated intervals to determine the numbers of CFU (details are provided in Materials and Methods). CFU results are means ⫾ SDs from 3 experiments. (A and B) Plankton-derived FOSC cells survived for up to 60 min in ML and 360 min in OP; (C and D) survival of biofilm-derived cells was notable for both the relatively higher numbers and the prolonged recovery at up to 240 min in ML and 360 min in OP.

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FIG. 4. Summary of experimental biofouling studies with FSSC evaluated by CFU method. Fungal cells (1 ⫻ 105 cells/ml) were inoculated into ML and OP solutions, and aliquots were withdrawn at the indicated intervals to determine CFU (details in Materials and Methods). CFU results are means ⫾ SDs from 3 experiments. (A and B) Plankton-derived FSSC cells survived for up to 60 min in ML and 360 min in OP; (C and D) survival of biofilm-derived cells was notable for both the relatively higher numbers and the prolonged recovery at up to 240 min in ML and 360 min in OP.

differences, all biofouling experiments were carried out without the neutralization step. Biofouling experiments (glass tubes versus lens cases). Results of glass tube versus lens case biofouling experiments obtained using ML are summarized in Table 2. There were no significant differences between glass tubes and lens cases using ML (P ⬎ 0.05). Therefore, all experiments were carried out in glass tubes. Biofouling experiments (CFU method). Results of MPS testing by traditional CFU methods are summarized in Fig. 3 and 4. Biofouling experiments using 1 ⫻ 105 plankton-derived cells/ml revealed that FOSC cells survived in ML for up to 60 min (Fig. 3A) and survived in OP for up to 360 min (Fig. 3B). There were no viable cells when two other MPSs (EQ and MP) were used (data not shown). Longer survival times were noted when biofouling experiments using 1 ⫻ 105 cells/ml of biofilm inoculum were used. FOSC biofilm cells survived in ML for up to 240 min (Fig. 3C) and survived in OP for up to 360 min (Fig. 3D). Similarly, for FSSC, the plankton-derived cells survived for up to 60 min in ML and up to 360 min in OP (Fig. 4A and B), while biofilm-derived cells survived for up to 240 min in ML and 360 min in OP (Fig. 4C and D). Similar to the results obtained with plankton-derived cells, the EQ and MP MPSs proved 100% efficient in killing biofilm-derived FSSC and FOSC cells (data not shown). Biofouling experiments (FC assay). The details for FOSC and FSSC exposed to various MPSs and tested by FC assay are

shown in Tables 3 and 4, respectively. The relative fluorescence of FOSC (MYC-158-05) cells treated with ML for various incubation times revealed that cells survived for up to 60 min and were nonviable at 240 min when plankton-derived cells were used (Fig. 5A), whereas biofilm-derived cells survived for up to 240 min (Fig. 5B). It is evident from the density plots that both plankton- and biofilm-derived cells comprised heterogeneous populations. Using two fluorochromes, four distinct populations could be discerned according to the emitted fluorescence: live cell subpopulation in the lower right quadrant (CMFDA⫹/Sytox Red⫺), dead cell subpopulation in the upper left quadrant (CMFDA⫺/Sytox Red⫹), injured cell subpopulation in the upper right quadrant (CMFDA⫹/Sytox Red⫹), and cell debris and cell subpopulation that did not retain any fluorochrome in the lower left quadrant (CMFDA⫺/Sytox Red⫺). As the last subpopulation was miniscule, further PCSs were recorded only for CMFDA⫹/Sytox Red⫺, CMFDA⫺/ Sytox Red⫹, and CMFDA⫹/Sytox Red⫹. A majority of plankton-derived cells appeared to be injured at as early as 60 min of ML exposure (CMFDA⫹/Sytox Red⫹); this changed to a complete loss of viability at 240 min (CMFDA⫺/Sytox Red⫹). Biofilm-derived cells retained viability for up to 240 min of exposure to ML (CMFDA⫹/Sytox Red⫺), and the proportion of injured cells (CMFDA⫹/Sytox Red⫹) was much lower than the proportion of plankton-derived cells exposed for similar time intervals. At 240 min, biofilm-derived cells mostly comprised injured cells, while

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TABLE 3. Details of percent FOSC cell subpopulations measured in a flow cytometer using CMFDA and Sytox Red after exposure to ML and OP Mean % cell subpopulation ⫾ SD Cell type, strain, and fluorescence characteristic

Planktonic cells MYC-158-06 CMFDA⫹/Sytox CMFDA⫹/Sytox CMFDA⫺/Sytox MYC-159-06 CMFDA⫹/Sytox CMFDA⫹/Sytox CMFDA⫺/Sytox MYC-398-06 CMFDA⫹/Sytox CMFDA⫹/Sytox CMFDA⫺/Sytox Biofilm cells MYC-158-06 CMFDA⫹/Sytox CMFDA⫹/Sytox CMFDA⫺/Sytox MYC-159-06 CMFDA⫹/Sytox CMFDA⫹/Sytox CMFDA⫺/Sytox MYC-398-06 CMFDA⫹/Sytox CMFDA⫹/Sytox CMFDA⫺/Sytox

ML

OP

15 min

30 min

60 min

240 min

15 min

30 min

60 min

360 min

Red⫺ Red⫹ Red⫹

44 ⫾ 5.3 55 ⫾ 4.9 1 ⫾ 0.4

3 ⫾ 4.5 91 ⫾ 4.1 3 ⫾ 0.6

1 ⫾ 0.6 92 ⫾ 3.1 3 ⫾ 0.7

0 1 ⫾ 0.2 96 ⫾ 5.6

62 ⫾ 7.9 32 ⫾ 4.5 6 ⫾ 2.3

30 ⫾ 8.7 50 ⫾ 9.8 20 ⫾ 4.8

10 ⫾ 4.6 30 ⫾ 7.6 60 ⫾ 8.9

8 ⫾ 1.1 13 ⫾ 4.5 79 ⫾ 10.1


48 ⫾ 7.1 50 ⫾ 6.9 2 ⫾ 0.9

15 ⫾ 5.6 82 ⫾ 4.2 3 ⫾ 0.9

6 ⫾ 0.4 22 ⫾ 5.6 72 ⫾ 4.9

0 14 ⫾ 1.1 86 ⫾ 7.9

65 ⫾ 4.6 29 ⫾ 4.7 6 ⫾ 2.5

33 ⫾ 7.5 49 ⫾ 4.8 18 ⫾ 2.3

15 ⫾ 2.3 16 ⫾ 5.6 69 ⫾ 4.2

7 ⫾ 1.4 11 ⫾ 4.1 82 ⫾ 4.4


38 ⫾ 7.1 59 ⫾ 6.7 3 ⫾ 1.0

18 ⫾ 5.4 78 ⫾ 5.5 4 ⫾ 0.8

9 ⫾ 2.3 42 ⫾ 4.5 49 ⫾ 4.4

0 38 ⫾ 4.4 62 ⫾ 5.6

45 ⫾ 4.5 53 ⫾ 3.0 2 ⫾ 0.7

24 ⫾ 3.1 59 ⫾ 3.5 17 ⫾ 1.8

17 ⫾ 2.1 68 ⫾ 5.1 15 ⫾ 2.5

9 ⫾ 2.1 9 ⫾ 2.5 79 ⫾ 6.4


50 ⫾ 6.3 15 ⫾ 2.1 6 ⫾ 1.3

47 ⫾ 3.5 24 ⫾ 1.4 9 ⫾ 1.5

22 ⫾ 2.5 48 ⫾ 4.7 18 ⫾ 2.5

9 ⫾ 1.5 70 ⫾ 2.0 19 ⫾ 1.5

62 ⫾ 4.5 30 ⫾ 3.0 8 ⫾ 0.9

45 ⫾ 3.5 22 ⫾ 3.0 33 ⫾ 5.9

26 ⫾ 2.0 26 ⫾ 4.5 58 ⫾ 7.3

14 ⫾ 2.0 17 ⫾ 3.0 70 ⫾ 8.4


78 ⫾ 5.8 22 ⫾ 4.5 0

52 ⫾ 6.0 16 ⫾ 3.5 32 ⫾ 2.1

36 ⫾ 4.7 36 ⫾ 4.4 28 ⫾ 1.4

22 ⫾ 2.4 10 ⫾ 1.5 78 ⫾ 8.7

68 ⫾ 9.6 27 ⫾ 6.2 5 ⫾ 1.0

39 ⫾ 4.6 33 ⫾ 2.3 28 ⫾ 2.5

23 ⫾ 2.1 36 ⫾ 4.5 41 ⫾ 8.7

11 ⫾ 4.6 15 ⫾ 7.2 72 ⫾ 7.6


66 ⫾ 6.3 30 ⫾ 4.2 4 ⫾ 2.1

48 ⫾ 3.5 36 ⫾ 2.1 16 ⫾ 4.6

25 ⫾ 5.9 36 ⫾ 1.7 39 ⫾ 1.1

11 ⫾ 1.7 20 ⫾ 5.8 69 ⫾ 7.9

71 ⫾ 4.9 18 ⫾ 3.9 11 ⫾ 1.8

43 ⫾ 8.9 36 ⫾ 9.5 21 ⫾ 7.8

22 ⫾ 4.8 45 ⫾ 8.7 33 ⫾ 7.7

9 ⫾ 4.5 12 ⫾ 6.4 85 ⫾ 7.9

smaller proportions of live and dead cell populations were still present. FC measurements of live, injured, and dead cells of FSSC (MYC-23-06) are depicted in Fig. 6. The relative fluorescence of the ML-treated, plankton-derived (Fig. 6A) or biofilm-derived (Fig. 6B) cells at various incubation times revealed that cells survived for up to 60 min and were killed at 240 min. This is illustrated as density plots, and the bar diagram shows the data obtained when 3 strains of FSSC were tested (mean ⫾ standard deviation [SD]). It is evident from the density plots that FSSC cells were different from FOSC cells, in that the majority of plankton-derived cells comprised a homogeneous population, while biofilm-derived cells were more heterogeneous. A majority of plankton-derived cells appeared to be injured at as early as 60 min of exposure (CMFDA⫹/Sytox Red⫹); this changed to a complete loss of viability at 240 min (CMFDA⫺/Sytox Red⫹). Biofilm-derived cells retained viability for up to 240 min of exposure to ML (CMFDA⫹/Sytox Red⫺), and the proportion of injured cells (CMFDA⫹/Sytox Red⫹) was much lower than the proportion of plankton-derived cells exposed for similar time intervals. At 240 min, biofilm-derived cells mostly comprised injured cells, while smaller proportions of live and dead cell populations were still present. Inoculum size and MPS efficacy. We further tested if the number of cells in a simulated contamination influences MPS efficacy. A 10-fold reduction of either plankton- or biofilmderived cells (1 ⫻ 104 cells/ml) resulted in relatively less survival of the cells for up to 30 min: 5 to 18% for FOSC and 2 to 12% for FSSC in MP and OP. A further reduction of the

inoculum (1 ⫻ 103 cells/ml) caused the least contamination of both ML and OP, with cells surviving for only 10 min: 1 to 3% for FOSC and 1 to 2% for FSSC (details not shown). Correlation of CFU and FC assay. Regression analysis of results obtained for viable cells measured by determination of the numbers of CFU and the rate of PCS positivity by CMFDA staining after exposure to ML and OP revealed an excellent correlation between CFU and FC measurements for various time intervals (r2 ⫽ 0.91 to 0.99; Fig. 7A to D). DISCUSSION We first developed a rapid FC assay for the analysis of MPS efficacy against Fusarium species. Second, we compared strains of FOSC and FSSC from keratitis patients to understand if there are noticeable differences in the susceptibility of these fungi to MPS. Third, we used plankton-derived and biofilmderived Fusarium cells to simulate either contaminated solutions or contaminated surfaces. Finally, we reproduced inefficient antifungal effects attributed to ML. We also report that two other brands of MPS have a high degree of efficacy against all forms of Fusarium cells even under shorter exposures to simulate inappropriate usage. In the current study, inappropriate disinfection applications were simulated by using 15- to 60-min intervals. This noncompliant use has been reported among contact lens users, as were confusing guidelines for appropriate usage (10, 16, 31). Previously, our data suggested that temporary survival of Fusarium in ML along with improper use of the lens cleaning regimen

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TABLE 4. Details of percent FSSC cell subpopulations measured in a flow cytometer using CMFDA and Sytox Red after exposure to ML and OP Mean % cell subpopulation ⫾ SD Cell type, strain, and fluorescence characteristic

Planktonic cells MYC-23-06 CMFDA⫹/Sytox CMFDA⫹/Sytox CMFDA⫺/Sytox MYC-288-06 CMFDA⫹/Sytox CMFDA⫹/Sytox CMFDA⫺/Sytox MYC-299-06 CMFDA⫹/Sytox CMFDA⫹/Sytox CMFDA⫺/Sytox Biofilm cells MYC-23-06 CMFDA⫹/Sytox CMFDA⫹/Sytox CMFDA⫺/Sytox MYC-288-06 CMFDA⫹/Sytox CMFDA⫹/Sytox CMFDA⫺/Sytox MYC-2998-06 CMFDA⫹/Sytox CMFDA⫹/Sytox CMFDA⫺/Sytox

ML

OP

15 min

30 min

60 min

240 min

15 min

30 min

60 min

360 min


18 ⫾ 1.2 78 ⫾ 5.9 2 ⫾ 0.25

6 ⫾ 0.9 90 ⫾ 11.2 4 ⫾ 0.75

3 ⫾ 0.5 92 ⫾ 0.5 6 ⫾ 0.75

0 0.5 ⫾ 0.1 95 ⫾ 5.25

32 ⫾ 4.0 36 ⫾ 2.5 6 ⫾ 2.5

26 ⫾ 2.8 48 ⫾ 1.0 20 ⫾ 1.0

11 ⫾ 1.0 30 ⫾ 0.5 59 ⫾ 0.5

11 ⫾ 0.5 19 ⫾ 0.5 81 ⫾ 0.5


44 ⫾ 6.4 52 ⫾ 4.6 4 ⫾ 0.3

18 ⫾ 1.3 82 ⫾ 6.6 3 ⫾ 0.5

9 ⫾ 1.0 22 ⫾ 5.4 72 ⫾ 4.4

0 14 ⫾ 2.3 86 ⫾ 11.1

42 ⫾ 5.0 48 ⫾ 3.2 9 ⫾ 1.3

31 ⫾ 3.6 56 ⫾ 1.8 25 ⫾ 2.2

21 ⫾ 3.6 44 ⫾ 1.9 65 ⫾ 3.3

14 ⫾ 1.8 15 ⫾ 1.1 71 ⫾ 2.6


46 ⫾ 5.5 44 ⫾ 5.0 10 ⫾ 1.3

28 ⫾ 4.5 62 ⫾ 2.4 10 ⫾ 0.5

15 ⫾ 2.0 35 ⫾ 2.0 50 ⫾ 4.4

0 19 ⫾ 1.5 81 ⫾ 11.1

32 ⫾ 6.3 57 ⫾ 3.1 11 ⫾ 1.2

28 ⫾ 4.2 62 ⫾ 5.5 10 ⫾ 2.9

11 ⫾ 4.1 36 ⫾ 5.7 53 ⫾ 4.8

6 ⫾ 1.5 7 ⫾ 0.6 87 ⫾ 1.2


59 ⫾ 3.3 32 ⫾ 3.6 9 ⫾ 1.2

55 ⫾ 3.1 34 ⫾ 2.7 11 ⫾ 0.9

28 ⫾ 2.2 38 ⫾ 1.4 44 ⫾ 3.1

14 ⫾ 1.1 36 ⫾ 1.2 50 ⫾ 5.1

62 ⫾ 2.9 27 ⫾ 1.5 11 ⫾ 1.9

52 ⫾ 2.8 38 ⫾ 1.4 10 ⫾ 1.9

15 ⫾ 2.9 61 ⫾ 1.7 24 ⫾ 0.4

9 ⫾ 1.4 29 ⫾ 4.6 62 ⫾ 2.2


52 ⫾ 2.3 42 ⫾ 3.6 6 ⫾ 1.2

45 ⫾ 1.8 25 ⫾ 1.5 30 ⫾ 3.6

32 ⫾ 1.3 20 ⫾ 0.9 48 ⫾ 4.8

18 ⫾ 1.8 6 ⫾ 0.6 76 ⫾ 4.9

52 ⫾ 2.2 38 ⫾ 1.9 10 ⫾ 1.5

42 ⫾ 1.8 31 ⫾ 0.9 27 ⫾ 1.1

18 ⫾ 2.6 51 ⫾ 1.9 31 ⫾ 0.7

11 ⫾ 2.5 19 ⫾ 2.3 70 ⫾ 2.6


62 ⫾ 1.2 30 ⫾ 2.6 8 ⫾ 0.5

57 ⫾ 2.5 35 ⫾ 2.2 18 ⫾ 1.1

40 ⫾ 2.2 18 ⫾ 1.5 42 ⫾ 1.4

22 ⫾ 2.3 8 ⫾ 1.0 70 ⫾ 3.5

56 ⫾ 2.2 35 ⫾ 1.9 9 ⫾ 1.5

48 ⫾ 1.8 30 ⫾ 0.9 22 ⫾ 1.1

12 ⫾ 2.6 52 ⫾ 1.9 36 ⫾ 0.7

12 ⫾ 2.5 16 ⫾ 2.3 72 ⫾ 2.6

could be a factor contributing to the keratitis outbreak (11). Under conditions simulating noncompliant use, ML showed reduced biocidal activity; however, MP retained a normal level of biocidal activity under similar conditions (19). The data in the present study revealed that both ML and OP had reduced activity against FOSC and FSSC, yet only ML has been implicated in the Fusarium keratitis outbreak. It is conceivable that these in vitro experiments do not replicate all possible conditions that led to the incidence of keratitis among contact lens users. Investigators from Singapore first reported that contact lens case hygiene practices were not optimal among infected individuals, and this was likely to have played a role in the Fusarium keratitis outbreak; their subsequent data found the strongest association only with the use of ReNu with MoistureLoc solution (16, 28). Furthermore, Chang et al. described that some other practices, such as storing of lenses in reused MPS, might have also facilitated the growth of Fusarium biofilms (8). In contrast, Ma et al. reported no significant association between poor hygiene practice and an ML-associated Fusarium keratitis outbreak in Hong Kong (20). The rapid results obtained with the FC assay are consistent with findings reported for testing of antifungal drugs against fungi pathogenic for humans (23–26). Indeed, FC remains the only technology that offers “quantitative estimation of antifungal effects in hours instead of days” (9). Importantly, we found an injured subpopulation of Fusarium cells treated with MPS which was intermediate between live and dead cells, and this information might be useful in further evaluations of MPSs. We suggest that this technology platform could be readily

adapted for evaluation of the antimicrobial activities of MPSs. In fact, previous studies have reported that FC provided rapid results that compared well with those of traditional plaque assay and direct microscopy when lens cleansers were evaluated for efficacy against Acanthamoeba castellanii (6, 17). In the standard methods for determining the susceptibilities of Fusarium species to various MPSs, only two types of population can be described, namely, live and dead cells, while the FC procedure distinguishes three populations, namely, live, dead, and injured cells. Therefore, not only is the FC method rapid, but it is also more informative, as it quantifies the contribution of each cell in the population. It may be relevant to add here that FC has been reported to be highly effective for rapid antifungal susceptibility testing and for determining the cell injury caused by antifungal drugs (9, 23, 24, 26). Our finding of the increased survival of biofilm-derived Fusarium cells reproduced some of the findings reported by Imamura et al. (14). These investigators developed an exquisite in vitro model of Fusarium as well as Candida albicans biofilms on various materials of contact lenses, and they reported that biofilms were more resistant to MPSs. Similar findings have been reported for Pseudomonas aeruginosa biofilms on contact lenses tested with various MPSs (30). Paradoxically, this scenario was reversed in a study of Staphylococcus aureus, for which planktonic cells proved more resistant than biofilms to MPSs (30). Clearly, biofilm-derived cells represent a unique cellular community that likely resists penetration and the antifungal effects of MPSs. Until recently, no information on the efficacy of MPSs against biofilm-derived fungal cells was avail-

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FIG. 5. Summary of experimental biofouling studies with FOSC evaluated by FC assay. Plankton-derived (A) or biofilm-derived (B) cells of strain MYC-158-06 were adjusted to final counts of 1 ⫻ 107 cells/ml. Ten microliters of this suspension was inoculated into 1.0 ml of ML. The mix was incubated unstirred at 25°C for various time intervals (15 to 240 min), and 200-␮l aliquots were removed intermittently. Cells were stained with CMFDA and Sytox Red and analyzed by a FACSCalibur apparatus (Becton Dickinson, Lincoln Park, NJ), and the results were plotted as density plots and histograms (means ⫾ SDs of three experiments). Both plankton- and biofilm-derived cells comprised heterogeneous populations. Remarkably, a majority of planktonic cells appeared to be injured by 60 min of exposure (CMFDA⫹/Sytox Red⫹); this changed to a complete loss of viability at 240 min (CMFDA⫺/Sytox Red⫹). Biofilm-derived cells retained viability with up to 240 min of exposure to ML (CMFDA⫹/Sytox Red⫺), and the proportion of injured cells (CMFDA⫹/Sytox Red⫹) was much lower than that for plankton-derived cells exposed for similar time intervals. At 240 min, biofilm-derived cells comprised a majority of injured cells, while smaller proportions of live and dead cell populations were still present.

able, because the recommended ISO method for testing MPS efficacy required use of only plankton-derived cells (4). Further, the biofilms formed on contact lenses vary in their architecture on the basis of the types of contact lenses used (14). It is not clear if biofilm architecture also affects MPS activity. The

scope of the present study was limited to a comparison of plankton-derived and biofilm-derived cells. Therefore, we elected to use a flow cell device, which is known to generate biofilms of uniform architecture. Future studies are warranted to discern any difference in MPS activity against fungal cells

FIG. 6. Summary of experimental biofouling studies with FSSC evaluated by FC assay. Plankton-derived (A) or biofilm-derived (B) cells of strain MYC-23-06 were adjusted to final counts of 1 ⫻ 107 cells/ml. Ten microliters of this suspension was used to inoculate 1.0 ml of ML. The mix was incubated unstirred at 25°C for various time intervals (15 to 240 min), and 200-␮l aliquots were removed intermittently. Cells were stained with CMFDA and Sytox Red and analyzed by a FACSCalibur apparatus (Becton Dickinson, Lincoln Park, NJ), and the results were plotted as density plots and histograms (means ⫾ SDs of three experiments). It is evident from the density plots that FSSC cells were different from FOSC cells, in that the majority of plankton-derived cells comprised a homogeneous population, while biofilm-derived cells were more heterogeneous. Remarkably, a majority of planktonic cells appeared to be injured with up to 60 min of exposure (CMFDA⫹/Sytox Red⫹); this changed to a complete loss of viability at 240 min (CMFDA⫺/Sytox Red⫹). Biofilm cells retained viability with up to 240 min of exposure to ML (CMFDA⫹/ Sytox Red⫺), and the proportion of injured cells (CMFDA⫹/Sytox Red⫹) was much lower than that of plankton-derived cells exposed to the similar time intervals. At 240 min, biofilm-derived cells comprised a majority of injured cells, while smaller proportions of live and dead cell populations were still present.

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FIG. 7. Comparison of results of biofouling experiments measured by CFU and FC assays. Regression analysis of results obtained by CFU assay and by FC assay of cells exposed to MPS. (A to D) An excellent correlation between CFU and FC measurements of viable cells after exposure to ML and OP solutions was observed (R2 ⫽ 0.91 to 0.99).

derived from biofilms formed on various contact lenses versus that against cells obtained from a flow cell device. Notably, members of FOSC survived longer than members of FSSC in two MPSs (ML and OP). This experimental finding was previously reported from our laboratory (11), and it might be relevant when one considers that F. oxysporum was reported in 59% of cases, while F. solani was reported in 9% of cases from some institutions (3, 8, 21). It is tempting to suggest that F. oxysporum is much more resistant to certain MPSs, and this probably explains its overwhelming presence among the isolates from the recent outbreak (3, 8, 21). An insight into this observed phenomenon has been provided in studies by Ahearn and colleagues, who reported that F. oxysporum strains showed greater penetration of contact lenses and survived better after treatment with MPS, while F. solani strains were easily removed (2, 35). However, these experimental findings must be reconciled with clinical studies, where other institutions have reported more FSSC in their keratitis patients (3, 16, 32) Clearly, in vitro experiments are limited in simulating all

of the conditions that predispose patients to Fusarium keratitis. In conclusion, the enigmatic outbreak of Fusarium keratitis in contact lens users could have resulted from a number of factors operating together or singly. The current ISO protocol is probably not adequate for testing the efficacy of lens cleaning solutions, as the method requires use of only one strain and also only plankton-derived cells. The use of a single laboratory strain and a single source of inoculum may provide a false sense of assurance that the disinfecting solution is effective; testing could be improved by the use of multiple clinical strains and, possibly, matching environmental isolates (5, 15). A provision for challenge against biofilmderived cells might provide an additional level of scrutiny of the efficacy of MPSs. Finally, the possibility that some formulations of MPSs are more robust in their efficacy even when they are used under conditions of noncompliance should be evaluated further.

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CONTACT LENS CLEANING SOLUTIONS AGAINST FUSARIUM ACKNOWLEDGMENTS

We acknowledge the excellent work of Kenneth Class of Immunology Core, Wadsworth Center, for flow cytometric analyses. We also thank Adriana Verschoor for critical comments on the manuscript. The study was supported in part by a research grant from Bausch and Lomb, Inc., to V.C. REFERENCES 1. Ahearn, D. G., et al. 2007. Attachment to and penetration of conventional and silicone hydrogel contact lenses by Fusarium solani and Ulocladium sp. in vitro. Cornea 26:831–839. 2. Ahearn, D. G., et al. 2008. Fusarium keratitis and contact lens wear: facts and speculations. Med. Mycol. 46:397–410. 3. Alfonso, E. C., et al. 2006. Insurgence of Fusarium keratitis associated with contact lens wear. Arch. Ophthalmol. 124:941–947. 4. Anonymous. 2001. ISO 14729. Ophthalmic optics-contact lens products-microbial rquirements and test method for products and regimes for hygienic management of contact lenses. International Organization for Standards, Geneva, Switzerland. 5. Boost, M., S. Lai, C. Ma, and P. Cho. 2010. Do multipurpose contact lens disinfecting solutions work effectively against non-FDA/ISO recommended strains of bacteria and fungi? Ophthalmic Physiol. Opt. 30:12–19. 6. Borazjani, R. N., L. L. May, J. A. Noble, S. V. Avery, and D. G. Ahearn. 2000. Flow cytometry for determination of the efficacy of contact lens disinfecting solutions against Acanthamoeba spp. Appl. Environ. Microbiol. 66:1057– 1061. 7. Bullock, J. D., R. E. Warwar, B. L. Elder, and W. I. Northern. 2008. Temperature instability of ReNu with MoistureLoc: a new theory to explain the worldwide Fusarium keratitis epidemic of 2004-2006. Trans. Am. Ophthalmol. Soc. 106:117–127. 8. Chang, D. C., et al. 2006. Multistate outbreak of Fusarium keratitis associated with use of a contact lens solution. JAMA 296:953–963. 9. Chaturvedi, V. 2008. Role of flow cytometry in medical mycology for antifungal testing, identification, and characterization. Curr. Fungal Infect. Rep. 2:143–148. 10. Donshik, P. C., W. H. Ehlers, L. D. Anderson, and J. K. Suchecki. 2007. Strategies to better engage, educate, and empower patient compliance and safe lens wear: compliance: what we know, what we do not know, and what we need to know. Eye Contact Lens 33:430–433. 11. Dyavaiah, M., et al. 2007. Molecular characterization, biofilm analysis and experimental biofouling study of Fusarium isolates from recent cases of fungal keratitis in New York State. BMC Ophthalmol. 7:1. 12. Hume, E. B., et al. 2009. Soft contact lens disinfection solution efficacy: clinical Fusarium isolates vs. ATCC 36031. Optom. Vis. Sci. 86:415–419. 13. Ide, T., D. Miller, E. C. Alfonso, and T. P. O’Brien. 2008. Impact of contact lens group on antifungal efficacy of multipurpose disinfecting contact lens solutions. Eye Contact Lens 34:151–159. 14. Imamura, Y., et al. 2008. Fusarium and Candida albicans biofilms on soft contact lenses: model development, influence of lens type, and susceptibility to lens care solutions. Antimicrob. Agents Chemother. 52:171–182. 15. Khan, S., et al. 2007. The role of CXC chemokine receptor 2 in Pseudomonas aeruginosa corneal infection. J. Leukoc. Biol. 81:315–318. 16. Khor, W. B., et al. 2006. An outbreak of Fusarium keratitis associated with contact lens wear in Singapore. JAMA 295:2867–2873. 17. Khunkitti, W., S. V. Avery, D. Lloyd, J. R. Furr, and A. D. Russell. 1997. Effects of biocides on Acanthamoeba castellanii as measured by flow cytometry and plaque assay. J. Antimicrob. Chemother. 40:227–233.

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